: The long term goal of this proposal is to understand the molecular antigen processing mechanism by which endogenously synthesized proteins yield precisely cleaved peptide/MHC class I complexes on the cell surface. The expression of these peptide/MHC complexes is essential for CD8+ cytotoxic T-cell immunity specific for tumors, intracellular pathogens, allogeneic tissue grafts and for autoimmunity. Failure to express peptide/MHC complexes is a major mechanism by which tumor cells and viruses such as herpes simplex virus, escape immune surveillance. Elucidating the molecular mechanism that allows endogenously synthesized proteins to be fragmented and displayed as peptide/MHC will also lead to understanding a key factor in determining epitope dominance, and thus improve design of vaccines for eliciting immunity against established tumors and intracellular pathogens. Novel T-cell based "lacZ" assays have been developed that permit quantitation and identification of processed peptides in the attomole range. Together with methods to express DNA constructs encoding defined antigenic precursors and for characterizing the processed peptide precursors and products in model cell lines the following problems will be addressed. (1) Whether naturally processed peptides arise as a consequence of cellular protein turn-over and/or from nascent proteins will be established by selective alteration of stability of antigenic precursor proteins via the N-end rule. (2) The influence of flanking residues on the generation of processed peptides will be determined by introducing specific mutations in model precursors and full-length proteins. The immunological consequences of altering the quantitative or qualitative outcome of antigen processing will be evaluated by measurement of T-cell responses to different dominant and sub-dominant peptides in these precursors. (3) The identity of processed peptide intermediates during the pre- and post-TAP step and their chaperones, that eventually yield the final precisely cleaved peptide products will be determined by analysis of HPLC fractionated cell extracts and novel assays. Finally, (4) we will generate and analyze new mutations in the antigen processing pathway. These genetic mutants are likely to reveal steps that occur during processing of endogenously synthesized precursors in living cells but may be inaccessible to conventional biochemical analysis.